Summary: Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the central and peripheral nervous systems and new ways to effectively control pain. The molecular research is performed using animal and in vitro, cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that send nerve fibers to skin and deep tissues and also connect with neurons in the dorsal spinal cord, which is the first site of synaptic information processing for pain. Our research has identified the DRG and spinal cord as loci of neuronal plasticity and altered gene expression in persistent pain states. The mechanisms of transduction of physical pain stimuli are investigated through examination of pathophysiological processes and molecules in damaged or inflamed peripheral tissue, or using reductionist preparations such as primary cultures or heterologous expression systems. Our goals are (1) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (2) to investigate mechanisms underlying human chronic pain disorders, and (3) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal by a translational research and human clinical trials program aimed at developing new molecular interventions for severe pain. The current approach, based on our studies of pain transduction through the vanilloid transient receptor potential ion channel (TRPV1), has resulted in a Phase I clinical trial of the TRPV1 agonist resiniferatoxin (RTX) as a new treatment for advanced cancer pain (<http://clinicalstudies.info.nih.gov/detail/A_2009-D-0039.html>). RTX activates TRPV1, which is an inflammation and heat-sensitive calcium/sodium ion channel that normally converts painful heat or low pH into nerve action potentials by opening the pore of the ion channel. The influx of ions depolarizes pain-sensing nerve endings and sends electrical signals to the spinal cord (which in turn sends the signals to the brain where we perceive pain). RTX is a potent capsaicin analog that props open the TRPV1 ion channel, causing calcium cytotoxicity and death of TRPV1 pain-sensing neurons, their axonal projections (i.e., the nerve fiber) or their nerve endings. RTX produces very effective pain control in pre-clinical models by several routes of administration. The central route involves administration into the cerebrospinal fluid around the spinal cord (intrathecal). We can also inject directly into sensory ganglia, both routes produce permanent effects because TRPV1-containing neurons or axons are killed. For cancer pain, after IRB approval of the clinical protocol and the Investigational New Drug application by the FDA, we treated several patients with severe pain from advanced cancer. Each patient is a unique case since the tumor presents and progresses differently in each one. Thus, multiple endpoints for determining efficacy are necessary. Peripheral routes of RTX administration include direct injection into skin, joints, nerve bundles or topically onto the cornea. Analgesia by these routes is temporary since nerve endings and axons can regrow. We are also in the process of generating new protocols for post-herpetic neuralgia and osteoarthritis (OA) The OA project is enabled by observations of strong efficacy and long duration of action upon intraarticular injection of RTX into the knee joints of dogs with OA. These results reinforce the objective of therapeutic use of vanilloid agonists for pain control and the translation to human patients. Early Translational Investigations: A new approach to using TRPV1 as an analgesic target involves the identification of small chemicals that act as positive allosteric modulators (PAMs) of TRPV1 activation by pH, vanilloid or endovanilloid lipid-like agonists. Our canine cancer pain study showed that TRPV1-expressing neurons are one of the most important transducers of painful stimuli, and understanding how these cells and this critical ion channel can be blocked, activated or sensitized is fundamental to understanding the initial step in pain transmission. My lab developed a high throughput calcium fluorescence assay for TRPV1 PAMs and we screened the Molecular Libraries small molecule collection at the NIH Chemical Genomics Center (NCGC). We discovered several new chemical compounds that enhance the activation of TRPV1 upon agonist binding to the orthosteric vanilloid site or by elevated H+ ions and suggest positive modulation of the TRPV1 ion channel open state. Secondary assays for specificity and following medicinal chemical modifications of the lead compounds have been, and are being, conducted. These studies have progressed to a third round of analog syntheses for one of the lead PAMs (NPT-32). Several new compounds based on NPT-32 are being optimized for in vivo stability. For evaluation of in vivo activity we developed two templates in the rat. One uses modulation of the capsaicin-induced decrease in core body temperature; the other uses potentiation of agonist-induced nerve terminal inactivation followed by assessment of analgesic activity. We demonstrated the MRS-1477 PAM exhibited positive activity in both tests. NPT-32 was tested for nerve terminal inactivation and it too was active. These results represent the first in vivo observations of TRPV1 positive allosteric modulation. The present studies reveal the existence of a new class of pharmacological agents for pain modulation and pain control. Our investigations support the idea that TRPV1 PAMs have the potential to yield novel, non-narcotic, selective, long-lasting analgesic agents that may be effective in acute, persistent, or chronic pain disorders. Basic Pain Mechanisms: Underlying the translational studies are investigations of molecular regulation of gene expression, neuronal function, behavior, and mechanisms of pain transduction. We are systematically investigating molecular alterations at the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord. One goal of this approach is to obtain a comprehensive molecular understanding of nociceptive process. Our studies reveal a complex, dynamic modulation of gene expression and cellular composition at all three steps. We have identified prominent roles for new, key molecules with distinct combinatorial patterns of expression among the three tissues. We are pursuing this work through several approaches in human post-mortem tissues and animal models. We have collected human trigeminal ganglia and samples of the medullary dorsal horn where the trigeminal makes its nociceptive connections. These tissues are being used for analysis the initial nociceptive circuit. We will also examine DRG and spinal cord of patients with defined pre-mortem pain disorders and make comparison to veterinary canine osteosarcoma patients. These animals are less extensively treated than human patients potentially mitigating confounding effects of anti-cancer medications. We use a method called RNA-seq to sequence all of the mRNAs in a given tissue or cell population. This method is comprehensive and quantitative. We also combine it with genetically manipulated mice and rats and fluorescence activated cell sorting (FACS) to obtain specific cell population for sequencing. These results have proven to be both compelling and informative, and allow us to define the pain transducing genetic complement of specific sensory ganglion neuronal populations and can lead to a deeper understanding of mechanisms that trigger and sustain chronic pain.